Research And Application Of Magnetic Controlled Microtissue Construction And Regulation In Tissue Regeneration

PURPOSEMagnetic control method,based on the magnetic nanoparticles(MNPs),provides a new way to construct multilayer cell sheet or microtissue.However,there are still some obstacles in tissue regeneration,such as the lack of a method to promote the differentiation of construction products and the problem of vascularization.Therefore,the mentioned problems are solved by preparing novel magnetic nanoparticles carrying growth factors,and constructing a magnetically controlled distraction system.METHODS1.We designed and prepared the graphene oxide coated ferromagnetic nanoparticles(n GO@Fe₃O₄ MNPs)and characterized their material and magnetic properties.Dental pulp stem cells(DPSCs)were cultured with MNPs to evaluate its biocompatibility.We co-incubated n GO@Fe₃O₄ MNPs with proteins to test their protein-loading ability.Then,BMP2 immobilized multilayer cell sheet(microtissue)was constructed by magnetic control,and the osteogenic effect was evaluated in vivo.Furthermore,we further constructed an osteochondral microtissue to evaluate the application prospect in complex tissue regeneration.2.Through molecular biology methods,the effect and mechanism of the magnetic field on Fe₃O₄ MNPs labeled cell function were researched.The proliferation and expression of angiogenesis-related factors were detected after the treating of magnetic controlled distraction method.Furthermore,the vascularization of magnetic controlled distraction microtissue was evaluated in vivo.RESULTS1.The n GO@Fe₃O₄ MNPs,with a diameter of about 10 nm,have excellent magnetic properties.The n GO@Fe₃O₄ MNPs did not affect cell proliferation and apoptosis.The MNPs were easily taken up by cells,which was beneficial to the accurate and controllable construction of multilayer cell sheets.Moreover,it can combine with protein reversibly,which was beneficial to the release of protein.The multilayer cell sheet immobilized with BMP2 was successfully prepared,and the osteogenic property was significantly promoted.Moreover,the complex microtissue with the ability of chondrogenic and osteogenic differentiation was constructed.2.Continuous magnetic controlled treatment promoted the expression angiogenesis-related genes of MNPs labeled cells,which may be related to the increase of NO synthesis induced by activation of Akt signaling pathway.The vertical,horizontal and multi directional magnetically controlled distraction of the microtissue changed the shape of microtissue,promoted cell proliferation and increased the expression of angiogenesis related factors.The multi-directional magnetically controlled distraction of the microtissue exerted good vascularization effect in vivo.CONCLUSIONSIn this study,the n GO@Fe₃O₄ MNPs carrying growth factor and the magnetically controlled distraction method,were used to improve the regulation of differentiation and vascularization of the multilayer cell sheet/microtissue.These results provided new ideas for the application of the controlled magnetic construction of microtissue in the biomedical field,especially in the field of tissue regeneration.